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 American Journal of Infection Control 49 (2021) 21−29

Contents lists available at ScienceDirect

American Journal of Infection Control

journal homepage: www.ajicjournal.org

Major Article

Systematic review with meta-analysis of the accuracy of diagnostic tests

for COVID-19
€ger Msc a, Mariana M. Fachi Msc a, Raquel O. Vilhena PhD b, Alexandre F. Cobre MSc a,
Beatriz Bo
Fernanda S. Tonin PhD a, Roberto Pontarolo PhD b,*
a , Curitiba, Brazil
Pharmaceutical Sciences Postgraduate Program, Health Sciences Sector, Federal University of Parana
b , Curitiba, Brazil
Department of Pharmacy, Federal University of Parana

Key Words: Objective: To collate the evidence on the accuracy parameters of all available diagnostic methods for detect-
SARS-CoV-2 ing SARS-CoV-2.
Coronavirus Methods: A systematic review with meta-analysis was performed. Searches were conducted in Pubmed and
Evidence Scopus (April 2020). Studies reporting data on sensitivity or specificity of diagnostic tests for COVID-19 using
Sensitivity
any human biological sample were included.
Specificity
Results: Sixteen studies were evaluated. Meta-analysis showed that computed tomography has high sensi-
tivity (91.9% [89.8%-93.7%]), but low specificity (25.1% [21.0%-29.5%]). The combination of IgM and IgG anti-
bodies demonstrated promising results for both parameters (84.5% [82.2%-86.6%]; 91.6% [86.0%-95.4%],
respectively). For RT-PCR tests, rectal stools/swab, urine, and plasma were less sensitive while sputum (97.2%
[90.3%-99.7%]) presented higher sensitivity for detecting the virus.
Conclusions: RT-PCR remains the gold standard for the diagnosis of COVID-19 in sputum samples. However,
the combination of different diagnostic tests is highly recommended to achieve adequate sensitivity and
specificity.
© 2020 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All
rights reserved.

INTRODUCTION
After the first case reports of an acute respiratory syndrome of
unknown etiology in the city of Wuhan, Hubei province (December
31, 2019), Chinese authorities identified a new coronavirus (SARS-
CoV-2) that causes the clinical disease COVID-19. The virus outbreak
spread quickly, significantly affecting all continents with more than
2 million people infected and thousands of deaths.1,2 Consequently,
nations are facing the overwhelming of health care systems and both
psychological and economic burdens. The lack of effective treatments
or prevention strategies has contributed toward the increase in the
number of cases, enhancing health care expenses with hospitaliza-
tions and palliative therapies. Additionally, there are limited diagnos-
tic tests available, which favors the growth of under-reporting of
cases.2,3
* Address correspondence to Roberto Pontarolo, PhD, Department of Pharmacy, Fed-
eral University of Parana, Av. Lothario Meissner, 632, 80210-170, Curitiba, Parana, Bra-
zil.
E-mail address: pontarolo@ufpr.br (R. Pontarolo).
Conflict of interest: None to report.
Patients report fever and cough, and most develop chest discom-
fort, difficulty in breathing or pneumonia, being clinically diagnosed
by imaging tests such as chest X-ray or computed tomography (CT).
CT equipment is widespread worldwide and the scan process is rela-
tively simples and quick, which enables rapid screening for suspected
patients. The typical findings of chest CT images for individuals with
COVID-19 are multifocal bilateral patchy ground-glass opacities or
consolidation with interlobular septal and vascular thickening in the
peripheral areas of the lungs. However, CT findings can change as the
disease progresses and these manifestations may also be compatible
with other viral pneumonias.4,5
In this context, the current gold standard for diagnosing COVID-19
is based on a molecular test of the reverse transcription polymerase
chain reaction (RT-PCR), aimed at detecting the RNA of the virus in
respiratory samples such as nasopharyngeal swabs or bronchial aspi-
rate.6 The real-time RT-PCR test provides a sensitive (the ability of
the test to correctly identify those patients with the disease 7,8) and
specific (the ability of the test to correctly identify those patients
without the disease 8) method to detect SARS-COV-2, with different
diagnosis protocols including sequences of target primers available
in the World Health Organization public database.6,9 However,

https://doi.org/10.1016/j.ajic.2020.07.011
0196-6553/© 2020 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Elsevier Inc. All rights reserved.
22 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo
researchers should be aware that this test can also give false nega-
tives if the amount of viral genoma is insufficient or if the correct
time-window of viral replication is missed.10 Although the COVID-19
incubation period is estimated to be 5 days, false negative results are
common within 7 days of infection. Additionally, RT-PCR process is
time-consuming and shortages in test kit supplies are common
worldwide—especially during the beginning of the epidemic
outbreak.11
Other simpler and rapid methods, such as serological testing of IgM
and IgG production in response to viral infection, can be used to
enhance the detection sensitivity and accuracy of the molecular test or
for screening purposes to assess antibody profiles in a large popula-
tion.12,13 Because antibodies are usually detected only 1-3 weeks after
the onset of symptoms, these tests are used to assess the overall infec-
tion rate in the community—including the rate of asymptomatic infec-
tions—or in remote areas where qPCR assays are not available.12,14
In this scenario, given the limitations of clinical diagnosis alone
(due to the similarity of the symptoms of COVID-19 infection with
those of other viruses) and the availability of different molecular and
serological tests with both technical advantages and disadvantages, it
is important to summarize the accuracy parameters of these methods
and investigate whether they are sufficiently specific or sensitive to
fit their role in practice. Few studies addressing the diagnostic perfor-
mance of tests for COVID-19 exist, with special focus only on com-
mercially assays available in a given country. In addition, according
to the different health care settings worldwide, different patterns on
testing may exist. For instance, the number of daily tests performed
per thousand people in Australia or in the United States is around
1.80, while in Europe is near 1.06 and in South America is lower 0.30
(https://ourworldindata.org/).
Thus, we aimed to perform a systematic review with meta-analy-
sis to gather evidence on the features of all available diagnostic test
for SARS-CoV-2, including parameters of sensitivity, specificity, posi-
tive and negative likelihood ratios and summary receiver operating
characteristic (SROC) curves, whenever possible.
METHODS
This study was conducted according to Preferred Reporting Items
for a Systematic Review and Meta-analysis of Diagnostic Test Accu-
racy Studies (PRISMA-DTA) statement and Cochrane Collaboration
recommendations.15,16
Search strategy
Systematic searches were conducted in Pubmed and Scopus with-
out limits of time-frame or language (last updated April 2020). The
search strategy included the following descriptors: “diagnostic,”
“test,” “assay,” “covid-19,” “sars-cov-2”and other terms combined
with Boolean operators AND and OR. The complete strategy is avail-
able in the supplementary material. Manual searches in the referen-
ces lists of included studies and in the gray literature (eg, Google
Scholar) were also performed.
Eligibility criteria
Titles and abstracts of retrieved articles were screened for eligibil-
ity. Relevant articles were read in full and those fulfilling inclusion
criteria had their data extracted. Two authors performed all the liter-
ature selection steps individually and then discussed the differences
with a third author.
Studies were included in this systematic review if they met all the
following eligibility criteria: (i) evaluation of any diagnostic method;
(ii) aimed at diagnosis of SARS-CoV-2 (COVID-19); (iii) using any
human biological sample; and (iv) reporting data on the accuracy of
the test (eg, sensitivity and/or specificity). We excluded studies pub-
lished in non-Roman characters.
Data extraction and bias assessment
The following data were independently extracted by 2 research-
ers: general study details (authors, year of publication, country of ori-
gin, study design, and sample size), methods, characteristics, and
diagnostic test results (true positive, TP; true negative, TN; false posi-
tive, FP; false negative, FN, sensitivity, specificity, and accuracy).
Two reviewers evaluated independently the risk of bias in each
study using the Diagnostic Precision Study Quality Assessment Tool
(QUADAS-2) recommended by the Cochrane Collaboration. The
assessment was performed using the Review Manager Software ver-
sion 5.3 17
Statistical analyses
The meta-analyses were performed according to the technique
and type of sample from each study (ie, by subgroups). Sensitivity,
specificity, positive likelihood ratio (PLR) and negative likelihood
ratio (NLR) were measured with a 95% confidence interval based on
the TP, TN, FP, and FN rates that were extracted from the included
studies.
Sensitivity, defined as the probability that a test result will be pos-
itive when the disease exists (true positive rate) was calculated
as = VP/(VP + FN). Specificity, defined as the probability that a test
result will be negative when the disease is not present (true negative
rate) was calculated as = VN/(VN + VP). The PLR is the ratio between
the probability of a positive test result given the presence of the dis-
ease and the probability of a positive test result given the absence of
the disease, that is = true positive rate/false positive rate, or expressed
as sensitivity/(1-specificity). The NLR is ratio between the probability
of a negative test result given the presence of the disease and the
probability of a negative test result given the absence of the disease,
that is = false negative rate/true negative rate, or expressed as (1-sen-
sitivity)/specificity.
SROC curves based on TP e FP rates were also built whenever pos-
sible to describe the relationship between test sensitivity and speci-
ficity. An area under the curve (AUC) close to 1 indicated a good
diagnostic performance of the test. All analyses were performed
using the Meta-DiscÓ version 1.4.7.
The heterogeneity of the studies was established by x2 analysis,
with inconsistency values (I2) greater than 50% being considered as
moderate heterogeneity, and I2 greater than 75% defined as high het-
erogeneity. Outcomes with I2 values greater than 50% were submit-
ted to sensitivity analysis (ie, hypothetical removal of studies).
RESULTS
A total of 1,089 articles were identified after duplicate removal. Of
these, 1,046 were excluded during the screening phase (title and
abstract reading), with 43 records being fully appraised. Sixteen stud-
ies were included finally in the systematic review.18-33 We were able
to include 14 trials in the quantitative analyses (meta-analysis): the
studies by Corman et al. 22 and Pfefferle et al. 33 did not address the
clinical application of the methods (Fig 1).
All studies included in this review (n = 2,297 patients) were pub-
lished in 2020, designed as retrospective observational cohorts, with
only one defined as a control-case study.32 Fourteen studies were
conducted in China,18-20,22-32 while Italy, 21 Netherlands,22 England,22
and Germany 33 contributed with one study each.
All studies presented a test group (patients diagnosed with
COVID-19), while only 6 trials used a control group (patients negative
for COVID-19 18,20,21,24,25,33). Patients from the test group were
 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo
Fig. 1. Flowchart of included studies.
previously diagnosed using the (gold standard) PCR technique. The
diagnostic methods were tested for the following samples: nasopha-
ryngeal swab,20,30,32 nasopharyngeal aspirate,20 throat swab,20,26,30,32
blood,21,24,25,27,28,30,32 saliva,20,27 sputum,20,30 urine,20,27,28,30 and
stool and rectal swabs.20,27,28,31 Table 1 summarizes the main charac-
teristics of the included studies.
Analytical parameters
Three studies evaluated the optimization of PCR parameters for
the detection of SARS-CoV-2.20,22,33 Chan et al. 20 developed and com-
pared the performance of 3 new essays of RT-PCR of RNA-dependent
RNA polymerase (RdRp)/helicase (Hel), spike (S) and nucleocapsid
(N) genes from SARS-CoV-2. Corman et al. 22 assessed several SARS-
related viral genomic sequences to design the best primer and probe
set. Pferfferle et al. 33 investigated a set of primer and probes, target-
ing the E gene, for use in an automated system (Cobas 6800 System;
see Table 2).
The genes E and RdRp were the most commonly used to detect
the COVID-19 virus, both with high analytical sensitivity (technical
limit of detection of 3.2 and 3.6 copies per reaction, respectively). The
detection of the gene N presented lower analytical sensitivity (8.3
copies per reaction). The probe used by these studies is indicated for
any SARS-CoV infection, including SARS-CoV-2. Process automation
by using the open channel of the Cobas 6800 systems significantly
increased the limit of detection.
Diagnostic accuracy of tests
Meta-analyses evaluating the parameters of accuracy (sensitivity,
specificity, PLR and NLR) of the reported tests were performed
(Supplementary Table S1), results are shown in Table 3.
23
Data on CT of the chest was reported by 6 trials.18,19,23,28,29,31
Meta-analysis showed this method to be sensitive (91.9%, 95% CI
89.8%-93.7%; heterogeneity between trials of I2 = 92.9%), however
with low specificity (25.1%, 95% CI 21.0%-29.5%, I2 = 32.8%; see Figure
S1 of the supplementary material for complete results).
Immunological tests (IgM and IgG) were evaluated in 5 trials as a
diagnostic method for COVID-19.21,24,25,27,32 The antibody dosage
was tested in whole blood samples,21,32 fingerstick blood,24
serum,24,25,27 and plasma.24 Overall, sensitivity and specificity were
higher when the combination of IgM and IgG antibodies was evalu-
ated (see Supplementary Figs S2, S3, and S4), reaching 84.5% (95% CI
82.2%-86.6%, I2 = 93.2%) and 91.6% (95% CI 86.0%-95.4%, I2 = 0%)
respectively. The SROC curves for the immunological diagnostic tests
are shown in Figure 2.
Seven studies addressed the diagnostic test by PCR.20,26-28,30-32
Meta-analyses were conducted according to the type of sample. Rec-
tal stool/swab (24.1%, 95% CI 16.7%-33.0%), urine (0.0%, 95% CI 0.0%-
3.7%), plasma (7.3%, 95% CI 4.1%-11.7%) were less sensitive for detec-
tion of COVID-19. Sputum (97.2%, 95% CI 90.3%-99.7%), saliva (62.3%,
95% CI 54.5%-69.6%), nasopharyngeal aspirate/swab and throat swab
(73.3%, 95% CI 68.1%-78.0%) were more sensitive for detecting the
virus (Fig S5 in supplementary material). Due to the limited number
of PCR studies with a control group, it was not possible to perform
statistical analyses on the parameters of specificity, PLR and NLR.
Only the studies by Xie et al. 28 and Yu et al. 30 tested the PCR method
in a control group. In both trials, specificity was 100% for stool, urine,
blood, nasal swab and throat swab samples, while throat swab and
sputum samples had specificities of 98.6% and 90.0%, respectively
(Table S2 in supplementary material). Sensitivity analyses were per-
formed for all meta-analyses with high heterogeneity results
(I2 > 50%); however, no additional differences were found compared
to the effects of the original analyses (data not shown).
24 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo

Table 1
Characteristics of the included studies.

Study Country No. of patients/ No. of control Reference method Evaluated method Sample Marker/gene
samples group patients/ (gene) type
samples

Ai, China 1,014 413 RT-PCR (ORF1ab; N) Chest CT Chest image -

2020 18
Long, China 36 - RT-PCR Chest CT Chest image -
2020 19
Cassaniti, 2020 Italy 50 60 RT-PCR (RdRp and E) LFIA Whole blood IgM/IgG
21

Chan, China 15/273 39 RT-PCR (RdRp-P2) RT-PCR Naso-pharyngeal Hel

2020 20 aspirate
Naso-pharyngeal swab
Throat swab
Saliva S
Sputum
Plasma
Urine N
Feces
Rectal swab
Corman, 2020 22 Nether-lands -* -* RT-PCR (RdRp) RT-PCR Sputum RdRp and E
England Nose swab
China Throat swab
Fecal
In vitro specific tran-
scribed RNA standards
SARS-CoV genomic RNA
from cell culture
Li, China 78 - RT-PCR (E) Chest CT Chest image -
2020a 23
Li, China 404 131 RT-PCR LFIA Whole blood or plasma IgM/IgG
2020b 24 (fingerstick or venous)
Liu, China 214 128 RT-PCR ELISA Serum IgM/IgG
2020 25
Pan, China 23 - RT-PCR or virus gene RT-PCR Throat swabs NR
2020 26 sequence highly Stool
homologous to Sputum
SARS-CoV-2
Pfefferle, 2020 Germany -* 110* - RT-PCR Swab E
33
In vitro transcribed RNA
of the E gene
of SARS-CoV-2
Purified RNA of SARS-
CoV (strain
Frankfurt-1)
To, China 23 - RT-PCR RT-PCR Saliva Hel
2020 27 Blood
Rectal swab
Urine
-
EIA Serum IgM/IgG
Xie, China 19 - RT-PCR RT-PCR Throat swab -
2020 28 Stool
Blood
Urine
Chest CT Chest image -
Xu, China 90 - RT-PCR Chest CT Chest image -
2020 29
Yu, China 76 - NR ddPCR and RT-PCR Nasal swab ORF1ab and N
2020 30 Throat swab
Sputum
Urine
Blood
Zhang, China 14 - RT-PCR NAT Stool -
2020 31 Oropharyngeal swab
Chest CT Chest image -
Zhao, China 173/535 - RT-PCR ELISA Plasma IgM/IgG
2020 32
E, envelope protein gene; ELISA, enzyme linked immunosorbent assay; Hel, helicase protein gene; LFIA, lateral flow immunoassay; N, nucleocapsid protein gene; NAT, nucleic acid
tests; NR, not reported; ORF1ab, open reading frame 1ab gene; RdRp, RNA-dependent RNA polymerase gene for SARS-CoV, SARS-CoV-2, and bat-SARS-related CoV; RdRp-P2, RNA-
dependent RNA polymerase specific gene for SARS-CoV-2; RT-PCR, real time reverse transcriptase polymerase chain reaction; S, spike protein gene.
*Samples were used only for the validation of the method (no clinical application).
 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo 25

Table 2
Analytical parameters reported by the included studies

Study Method Probe RNA Gene target LoD

RNA copies/reaction (CI)

Chan, RT-PCR specific for SARS-CoV RdRp/Helicase 11.2 (7.2-52.6)

2020 20 RT-PCR specific for SARS-CoV Spike gene NA
RT-PCR specific for SARS-CoV N gene 21.3 (11.6-177.0)
RT-PCR specific for SARS-CoV-2 RdRp gene NA
Corman, 2020 22 RT-PCR specific for SARS-CoV E gene 5.2 (3.7-9.6)
(new method)
RT-PCR specific for SARS-CoV RdRp gene 3.8 (2.7-7.6)
(new method)
RT-PCR TaqMan Fast specific for SARS-CoV E gene 3.2 (2.2-6.8)
RT-PCR TaqMan Fast specific for SARS-CoV RdRp gene 3.7 (2.8-8.0)
RT-PCR specific for SARS-CoV-2 E gene 3.9 (2.8-9.8)
(new method)
RT-PCR specific for SARS-CoV-2 RdRp gene 3.6 (2.7-11.2)
(new method)
Pfefferle, RT-PCR specific for SARS-CoV-2 E gene 275.72 (NR)
2020 33
CI, confidence interval 95%; LoD, limit of detection; NA, not applied; NR, unreported.

Table 3
Meta-analysis of the parameters of accuracy for the different diagnostic techniques

Technique Sample No. of studies Sensitivity Specificity PLR NLR

(95% CI) (95% CI) (95% CI) (95% CI)

Computed tomography - 6 18,19,23,28,29,31 0.919 0.251 1.194 0.301

(0.898-0.937) (0.210-0.295) (0.936-1.525) (0.043-2.124)
I2 = 92.9% I2 = 32.8% I2 = 56.2% I2 = 71.9%
Immunological test Blood, serum, plasma 4 21,24,25,32 0.845 0.916 7.604 0.170
(IgM and IgG) (0.822-0.866) (0.860-0.954) (3.903-14.817) (0.041-0.697)
I2 = 93.2% I2 = 0.0% I2 = 12.8% I2 = 97.0%
Immunological test Blood 3 21,24,32 0.863 (0.833-0.888) 0.907 (0.848-0.948) 8.618 (5.219-14.231) 0.146 (0.021-1.028)
(IgM and IgG) I2 = 96.3% I2 = 0.0% I2 = 0.0% I2 = 99.0%
Immunological test Serum 2 24,25 0.82 (0.78-0.85) - - -
(IgM and IgG) I2 = 35.8%
Immunological test (IgM) Blood, serum, plasma 5 21,24,25,27,32 0.770 0.933 7.295 0.211
(0.745-0.795) (0.886-0.965) (3.403-15.641) (0.067-0.666)
I2 = 89.9% I2 = 18.5% I2 = 96.1% I2 = 96.1%
Immunological test (IgM) Blood 3 21,24,32 0.788 (0.754-0.819) 0.931 (0.882-0.964) 8.390 (3.367-20.905) 0.274 (0.072-1.043)
I2 = 94.8% I2 = 43.3% I2 = 24.0% I2 = 98.0%
Immunological test (IgM) Serum 3 24,25,27 0.743 (0.701-0.782) - - -
I2 = 73.1%
Immunological test (IgG) Blood, serum, plasma 5 21,24,25,27,32 0.694 0.694 25.626 0.378
(0.666-0.721) (0.666-0.721) (7.131-92.087) (0.128-1.111)
I2 = 90.9% I2 = 0% I2 = 18.0% I2 = 98.6%
Immunological test (IgG) Blood 3 21,24,32 0.661 (0.623-0.698) 0.988 (0.958-0.999) 26.981 (6.240-116.655) 0.377 (0.128-1.113)
I2 = 94.5% I2 = 0.0 I2- 27.3% I2 = 98.6%
Immunological test (IgG) Serum 2 25,27 0.739 (0.696-0.779) - - -
I2 = 80.7%
PCR Stool, feces, rectal swabs 4 20,27,30,31 0.241 - - -
(0.167-0.330)
I2 = 82.6%
PCR Urine 4 20,27,28,30 0.000 - - -
(0.000-0.037)
I2 = 0.0%
PCR Blood 3 21,27,28 0.073 - - -
(0.041-0.117)
I2 = 85.9%
PCR Nasopharyngeal 4 20,26,30,32 0.733 - - -
aspirate, nasopharyngeal (0.681-0.780)
and throat swab I2 = 87.5%
PCR Sputum 2 20,30 0.972 - - -
(0.903-0.997)
I2 = 48.3%
PCR Saliva 2 20,27 0.623 - - -
(0.545-0.696)
I2 = 92.2%
CI, confidence interval; I2, inconsistency; NLR, negative likelihood ratio; PCR, Polymerase chain reaction; PLR, positive likelihood ratio.
26 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo
Fig. 2. SROC curves obtained for immunological tests.
Quality assessment
Studies were rated as being of moderate overall methodological
quality according to QUADAS-2 (see Fig. 3 and 4). The studies by
Chan et al. 20 and Pfefferle et al. (29) were not evaluated given the
lack of clinical application of the tests (ie, only the analytical perfor-
mance of the methods was assessed).
Around one-quarter of the trials (26%) did not describe the meth-
ods of patient selection, and almost half (46%) included previously
diagnosed patients, which may enhance the risk of bias. However,
the majority of the patients included matched the review question
and were likely to be diagnosed with the evaluated tests (ie, no major
concerns for the applicability domain). Overall, 80% of the studies
properly reported both index and reference standard tests and how
they were conducted and interpreted. Only 3 studies (20%) properly
reported the interval between tests, whether patients received differ-
ent index or standard assays, and the complete statistical analyses
performed, thus being judged as having low risk of bias for the flow
and timing domain. The remaining studies were classified as with an
unclear risk of bias for this domain.
DISCUSSION
To our knowledge, this is the first systematic review with meta-
analysis to collate the available evidence on the accuracy parameters
of different diagnostic methods (clinical, molecular, and serological)
for the detection of SARS-CoV-2 in different samples, including blood,
nasopharyngeal swab, sputum, saliva, urine and feces. We were also
able to evaluate qualitatively the main analytical parameters
reported in the molecular techniques.
The development of new molecular techniques depends on the
knowledge of the proteomic and genomic composition of the virus or
of changes in the hosts’ protein expressions during and after infec-
tion.34 Genome sequencing is important for researchers to design pri-
mers and probes for PCR and other molecular tests. The SARS-CoV-2
virus has a single-stranded, positive RNA genome of approximately
30,000 nucleotides in length that encodes 27 proteins, including an
RdRP and 4 structural proteins: surface glycoprotein (S), envelope
protein (E), matrix protein (M) and nucleocapsid protein (N; 34,35. In
the past months, different RT-PCR kits for the detection of SARS-CoV-
2 have been developed, being able to amplify a small amount of viral
genetic material in a sample.36 In this technique, the RNA of the virus
is reverse-transcribed into complementary DNA strands (cDNA),
whose specific regions are amplified. The process usually involves 2
main steps: sequence alignment and primer design, and assay opti-
mization and testing, especially because this method requires several
temperature changes for each cycle using thermocycling
equipment.34
We found that in trials evaluating the RdRp/Hel gene, there were
no cross-reactions with other pathogenic coronaviruses and human
respiratory pathogens in cell culture or clinical samples. On the other
hand, the specific SARS-CoV-2 (RdRp) gene reacted with SARS-CoV in
 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo
Fig. 3. Methodological quality of the included studies (individual assessment).
cell culture.20 It is important to avoid the use of genes that could
potentially cause false-negative results. Assays designed as 2-target
systems, with a primer universally detecting several coronaviruses
(including SARS-CoV-2), and a second set of primers specifically
detecting SARS-CoV-2, are the most suitable to obtain lower LoDs.22
However, the use of different reagents, primer/probe concentrations,
and cycling conditions may limit the sensitivity of the test.20,34 In this
context, Pfefferle et al. 33 proposed an automated solution for molec-
ular diagnosis, including the management of a large volume of sam-
ples. The system used in this study (Cobas 6800 System) fully
automates the extraction, purification, amplification, and detection of
nucleic acids. Nonetheless, the analytical performance of the method
Fig. 4. Summary of the methodological quality of the included studies.
27
was lower compared to conventional techniques.20,22 This may arise
partly from differences in the determination of LoD among studies.
While in conventional methods the target RNA is added manually to
the reagent mix for amplification, in automated systems the control
(purified RNA) is inserted into the samples and passes through the
entire workflow of the device, including extraction and purification.33
Our meta-analyses demonstrated that among all methods, the PCR
technique using sputum samples was the most sensitive method for
diagnosing COVID-19. In contrast, this same technique applied to
other samples (eg, urine, blood, stool, feces, and rectal swabs) showed
the worst sensitivity results. Considering that the nucleic acid test is
the main diagnostic test for this infection,37,38 the choice of the type
of sample is an important step for successful diagnosis. Comparison of
the meta-analysis results from different clinical specimens clearly
demonstrated that respiratory samples are the most suitable for
achieving higher sensitivity rates. These results corroborate with the
CDC recommendations, which state that initial tests for the diagnosis
of SARS-CoV-2 should prioritize the collection of respiratory sam-
ples.37,38 One aspect that should not be disregarded is the fact that the
presence of the virus in the different biological samples is related to
the period of collection, that is, to the clinical course of the disease.
Yet, even at low concentrations these other specimens, as well as
respiratory fluids, may be involved in the transmission of the disease.
A recent study showed that SARS-CoV-2 may exist in children's gas-
trointestinal tract for a longer time than in the respiratory system.39
Our study also demonstrated that CT was the second most sensi-
tive test. SARS-CoV-2 is known to infect primarily the respiratory sys-
tem, causing inflammation, interstitial damage, changes in the
parenchyma, and cell death.5 Thus, the manifestations in CT of the
chest have been considered as a very important strategy for supple-
mentary diagnosis in view of the limitations of other techniques,
such as the case of false-negative results with RT-PCR.40 However,
this method has low specificity and a low PLR compared to immuno-
logical tests, which may hamper its isolated use in clinical practice.
This can be explained by the chest imaging findings being due to
other viral infections.
Regarding immunological tests, higher sensitivity, specificity and
better NLR were obtained when the total antibodies were evaluated.
On the other hand, the best PLR result was presented by the IgG
immunological test. The global assessment of the value of this diag-
nostic test also demonstrated it to have the highest AUC value in the
SROC curve (0.9992, Q* = 0.9935). Nonetheless, both IgM alone and
combined with IgG showed high AUC values (0.9601, Q* = 0.9046;
0.9581, Q* = 0.9018, respectively), similarly to what was reported by
Castro et al.,41 indicating a high level of accuracy for immunological
tests in the diagnosis of COVID-19. The antibodies researched in these
tests refer to structural antigenic proteins of SARS-CoV-2, such as
spike (S) and nucleocapsid (N) proteins, which have been identified
as the most relevant in the development of serological assays for the
diagnosis of the infection.14 Usually, the body's immune response to
28 €ger et al. / American Journal of Infection Control 49 (2021) 21−29
B. Bo
a pathogen takes 1-2 weeks to occur. In this context, the use of sero-
logical tests for detection in the initial/acute phase of the disease can
be challenging. A recent study showed that IgM and IgG seroconver-
sion can occur simultaneously or sequentially in COVID-19, and that
antibody titers reach a plateau after 6 days.19 In addition, a meta-
analysis of the accuracy of diagnostic tests marketed in Brazil, taken
from manufacturers' data, showed a range of 10%-40% false-negative
results for detection of SARS-CoV-2 IgM in the acute phase in 8 evalu-
ated tests.41 However, immunological tests have a quick turnaround
time and relatively low costs (around £6 per test or USD$ 8-10),42
which may represent an important strength for this method, given
the shortages of RT PCR and its higher price (around £30/test, but
may range from USD$ 25 to 100). Additionally, the participation of
multiple manufacturers in the market can potentially scale the
immunological tests to millions of people per day due to their simpler
design. This may especially help to improve the detection of the virus
in health care settings where resources are more limited, such as in
developing countries.42,43
Our study has some limitations. The included studies differ in terms
of size, risk of bias, and external validity. We are aware of potential
introduction of bias caused by studies of poor methodological quality.
We found high heterogeneity rates among trials, probably cause by
some differences in the methods, patient characteristics, and samples
used. However, we tried to avoid systematic errors by performing sen-
sitivity analyses, which do not demonstrate significant differences from
the original analyses. The eligibility criteria and description of the par-
ticipants is crucial, as the test is only valid under similar circumstances.
Sensitivities, specificities, TP, and TN were compared, but these statis-
tics depend on the populations studied, the reference tests used, and
the specific function of the test. Studies were judged as being of moder-
ate methodological quality, with few concerns regarding the applicabil-
ity of the methods used. The low reporting quality of some studies
hampered the performance of further analyses.
CONCLUSIONS
RT-PCR remains the gold standard for the diagnosis of COVID-19 in
sputum samples. However, depending on the type of sample and stage
of the disease, other methods are preferable. A combination of clinical,
molecular, and serological diagnostic tests is highly recommended to
achieve adequate sensitivity and specificity. Automated assays for
molecular diagnosis using a 2-target system for detecting SARS-CoV-2
should be used whenever possible to enhance analytical performance.
Acknowledgments
The authors express their gratitude for research funding to the
CAPES (Brazilian Federal Agency for Support and Evaluation of Grad-
uate Education within the Ministry of Education of Brazil)—Finance
Code 001.
SUPPLEMENTARY MATERIALS
Supplementary material associated with this article can be found
in the online version at https://doi.org/10.1016/j.ajic.2020.07.011.
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